It is proposed to establish with greater precision the H'/site and H'/ATP ratios of mitochondrial electron transport and ATP hydrolysis/synthesis, as they occur in intact mitochondria isolated from different animal tissues. Similar approaches will be taken to elucidation of the H ion /ATP ratios in bacteria and in submitochondrial vesicles. Once these stoichiometric ratios are firmly established we shall attempt to correlate these into a more quantitative description of respiratory energy transduction in mitochondria and the possible participation of an electrochemical gradient of protons as an obligatory energy-rich intermediate state during oxidative phosphorylation. We also expect to begin studies of the specific carriers in the respiratory chain that participate in ejecting H ion from the mitochondrial matrix into the cytosol during mitochondrial electron transport. These studies are in total aimed at a molecular description of the H ion-pumping process coupled to electron transfers. We plan to identify the major factors that regulate the retention and release of Ca 2 ions from mitochondria. In particular, we shall investigate the role of mitochondrial bound Mg 2 ions and the role of the oxidation-reduction state of specific electron carriers in the respiratory chain, particularly NAD and NADP, in triggering Ca 2 ion release. Since mitochondria from normal and tumor cells differ strikingly in their capacity for Ca 2 ion retention, we expect to compare the known factors leading to Ca 2 ion release in the two types of mitochondria. We also hope to investigate further the transport mechanisms that function in the transport of ADP into mitochondria in exchange for ATP coming out, as well as those participating in the transport of pyruvate, dicarboxylates, and other metabolites involved in those metabolic cycles whose enzymatic steps are divided between the cytosol and mitochondrial compartment.